Eduard Y. Kostetsky

Thermotropic behavior of major phospholipids from marine invertebrates: changes with warm-acclimation and seasonal acclimatization.

The crystal-liquid crystal-isotropic melt phase transitions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from muscle tissue of five species (actinia Metridium senile fimbriatum, mussel Crenomytilus grayanus, sea-urchin Strongylocentrotus intermedius, starfish Distolasterias nipon and the ascidian Halocynthia aurantium) of marine invertebrates, collected in winter at 0 degrees C and then acclimated to 18.5 degrees C for 5 days, were studied by differential scanning calorimetry and polarising microscopy. To elevate temperature from 0 to 18.5 degrees C, we used the rate of 4.5 degrees C/h. Although phase transitions of both phospholipids from animals collected in summer occurred already at temperatures below -1.7 oC (minimal temperature of seawater in winter), compensatory mechanisms resulted in a decrease by 29-43 oC in the phase transition temperature of PE in winter. Thermotropic behavior of PCs changed in various trends. However, the total heat of their phase transitions always decreased in winter compared with summer. For all species, except the mussel, the time of warm-acclimation was insufficient to adjust the thermotropic behavior of either phospholipid. Nevertheless, the unsaturation index decreased to achieve summer values, due primarily to decreased proportions of eicosapentaenate and docosahexaenate. The accumulation of arachidonate, during warm-acclimation, might be connected to the signalling properties of n-6 eicosanoids. Absence of effective homeoviscous mechanisms suggests that most of the studied marine invertebrates have very limited capacity to survive an acute temperature elevation, e.g. at the appearance of thermal currents.

Tubular immunostimulating complex based on cucumarioside A2-2 and monogalactosyldiacylglycerol from marine macrophytes

BackgroundThere is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity.ResultsThe cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times.ConclusionsWe propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.

Seasonal changes in thermotropic behavior of phosphatidylcholine and phosphatidylethanolamine in different organs of the ascidian Halocynthia aurantium

Differential scanning calorimetry and polarising microscopy were used to investigate the crystal-liquid crystal-isotropic melt phase transitions of phosphatidylcholine (PC), and phosphatidylethanolamine (PE), isolated from muscles, gill pouches, gonads and digestive glands of Halocynthia aurantium, collected in summer and winter. We also analyzed the fatty chain composition of these phospholipids. In summer, the crystalline to liquid crystalline phase transitions of PC and PE from different organs were more co-operative than in winter. Their peak maximum temperatures were close and temperature ranges overlapped for summer samples. Peak maximum temperatures of winter samples decreased sharply, by 18-27 degrees C for PC and by 10-44 degrees C for PE, respectively, depending on the organ. Total heat changes of transitions also decreased. Thermograms were completely located at temperatures below -1.7 degrees C (minimal temperature of seawater in winter). In contrast to summer samples, peak maximum temperatures for PC and PE in winter differed significantly, (by 14-30 degrees C depending on organ), while the temperature ranges of their transitions still showed considerable overlap. Simultaneously, the temperature ranges of the liquid crystalline to isotropic phase transitions decreased. The main reason for changes in thermotropic behavior of phospholipids seems to be the decrease of saturated/unsaturated ratios. The existence of stable and thermoadaptative labile phospholipid pools in the membrane structure is proposed. The relationship of these transitions to low- and high-temperature adaptation is discussed.

Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa

Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage.

Purification and structural stability of white Spanish broom (Cytisus multiflorus) peroxidase

New plant peroxidase has been isolated to homogeneity from the white Spanish broom Cytisus multiflorus. The enzyme purification steps included homogenization, NH(4)SO(4) precipitation, extraction of broom colored compounds and consecutive chromatography on Phenyl-Sepharose, HiTrap™ SP HP and Superdex-75 and 200. The novel peroxidase was characterized as having a molecular weight of 50 ± 3 kDa. Steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the structural stability of C. multiflorus peroxidase (CMP) at pH 7.0. Thus changes in far-UV CD corresponded to changes in the overall secondary structure of enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. It is shown that the process of CMP denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N ⟶ kD, where k is a first-order kinetic constant that changes with temperature following the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Kinetics of Spanish broom peroxidase obeys a Ping-Pong Bi–Bi mechanism with competitive inhibition by substrates

In plants, adverse conditions often induce an increase in reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). H2O2 is reduced to water, and thus becomes detoxified by enzymes such as Cytisus multiflorus peroxidase (CMP). Here, the steady-state kinetics of the H2O2-supported oxidation of different organic substrates by CMP was investigated. Analysis of the initial rates vs. H2O2 and reducing substrate concentrations proved to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters [Formula: see text] , [Formula: see text] , kcat, [Formula: see text] , [Formula: see text] and of the microscopic rate constants, k1 and k3, of the shared three-step catalytic cycle of peroxidases.

On the possibility of lipid-induced regulation of conformation and immunogenicity of influenza a virus H1/N1 hemagglutinin as antigen of TI-complexes

The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. MGDG is a lipid matrix for the protein antigen incorporated in the TI-complex. The aim of the present work was to study the influence of MGDGs from different macrophytes on conformation and immunogenicity of the secreted recombinant uncleaved hemagglutinin monomer (HA0S) of influenza A virus H1/N1. Differential scanning calorimetry, fluorescence spectroscopy and circular dichroism showed a dependence of the conformational changes of HA0S on the microviscosity of MGDG. The most viscous MGDG from Zostera marina induced the strongest rearrangements in protein conformation. Immunization of mice with HA0S within TI-complexes comprising different MGDGs resulted in an approximately 2-fold increase of the levels of anti-HA0S antibodies and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared with those induced by HA0S alone. TI-complexes based on MGDG from Z. marina stimulated the maximal production of GM-CSF. However, humoral immune response (anti-HA0S antibodies), unlike cell-mediated immune response (GM-CSF), did not depend on the physicochemical properties of MGDGs. It is assumed that this is due to the different localization and conformational lipid sensitivity of the HA0S regions, which are responsible for these types of immune responses.

The effects of triterpene glycosides and phospholipids from marine invertebrates in the composition of tubular immunostimulating complexes on the immunogenicity of human serum albumin

To study the adjuvant activity and to optimize tubular immunostimulating complexes (TI complexes) with respect to the soluble antigen, an investigation was performed on cucumariosides and their fractions, i.e., individual triterpene glycosides that were obtained from the holothurian Cucumaria japonica, as well as on phospholipids from the sea urchin Strongylocentrotus intermedius and the starfish Distolasterias nippon included in the composition of TI complexes in order to assess their effects on the immunogenicity of human serum albumin (HSA). Generally, TI complexes showed a moderate adjuvant activity with respect to HSA; this activity can be adjusted by substitution of a cucumarioside and/or a polar lipid in the composition of the TI complex. TI complexes based on monogalactosyldyacylglycerol (MGDG) from the sea alga Ulva lactuca and cucumariosides A2-2 and A4-4 maximally stimulated anti-HSA antibody production in mice. Substitution of MGDG for phosphatidylcholine (PC) from the starfish D. nippon with an increased (compared with other investigated phospholipids) ratio of n-3/n-6 polyunsaturated fatty acids did not result in a change in the immunostimulating activity of the TI complex. However, the use of other phospholipids led to a decrease in the adjuvant activity of the TI complex, compared with that of the TI complex based on PC from D. nippon and cucumarioside A2-2. The greatest fluctuations in the contents of cytokines IL-6, IL-12, IFN-γ and GM-CSF depending on the composition of the TI complexes indicate the possibility of regulation of the T-cell immune response. The values of hematological parameters were normal.

Thermal stability of matrix protein from Newcastle disease virus.

The thermal stability of the matrix protein (M protein) of Newcastle disease virus (NDV) has been investigated using high-sensitivity differential scanning calorimetry (DSC) at pH 7.4. The thermal folding/unfolding of M protein at this pH value is a reversible process involving a highly cooperative transition between folded and unfolded monomers with a transition temperature (Tm) of 63 °C, an unfolding enthalpy, ΔH(Tm), of 340 kcal mol(-1), and the difference in heat capacity between the native and denatured states of the protein, ΔCp, of 5.1 kcal K(-1) mol(-1). The heat capacity of the native state of the protein is in good agreement with the values calculated using a structure-based parameterization, whereas the calculated values for the hypothetical fully-unfolded state of the protein is higher than those determined experimentally. This difference between the heat capacity of denatured M protein and the heat capacity expected for an unstructured polypeptide of the same sequence, together with the data derived from the heat-induced changes in the steady-state fluorescence of the protein, indicates that the polypeptide chain maintains a significant amount of residual structure after thermal denaturation.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis

BACKGROUND Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.